The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs
2018; Springer Nature; Volume: 20; Issue: 1 Linguagem: Inglês
10.15252/embr.201846588
ISSN1469-3178
AutoresLilli Olgeiser, Carl Haag, Susan Boerner, Jernej Ule, Anke Busch, Janine Koepke, Julian König, Michael Feldbrügge, Kathi Zarnack,
Tópico(s)RNA modifications and cancer
ResumoArticle14 December 2018free access Transparent process The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs Lilli Olgeiser Lilli Olgeiser Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Carl Haag Carl Haag Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Susan Boerner Susan Boerner Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Jernej Ule Jernej Ule orcid.org/0000-0002-2452-4277 The Francis Crick Institute, London, UK Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Anke Busch Anke Busch Institute of Molecular Biology gGmbH, Mainz, Germany Search for more papers by this author Janine Koepke Janine Koepke Medical Clinic II (Molecular Pneumology), Excellence Cluster Cardio-Pulmonary System, Justus Liebig University of Gießen, Gießen, Germany Search for more papers by this author Julian König Julian König Institute of Molecular Biology gGmbH, Mainz, Germany Search for more papers by this author Michael Feldbrügge Corresponding Author Michael Feldbrügge [email protected] orcid.org/0000-0003-0046-983X Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Kathi Zarnack Corresponding Author Kathi Zarnack [email protected] orcid.org/0000-0003-3527-3378 Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Lilli Olgeiser Lilli Olgeiser Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Carl Haag Carl Haag Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Susan Boerner Susan Boerner Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Jernej Ule Jernej Ule orcid.org/0000-0002-2452-4277 The Francis Crick Institute, London, UK Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Search for more papers by this author Anke Busch Anke Busch Institute of Molecular Biology gGmbH, Mainz, Germany Search for more papers by this author Janine Koepke Janine Koepke Medical Clinic II (Molecular Pneumology), Excellence Cluster Cardio-Pulmonary System, Justus Liebig University of Gießen, Gießen, Germany Search for more papers by this author Julian König Julian König Institute of Molecular Biology gGmbH, Mainz, Germany Search for more papers by this author Michael Feldbrügge Corresponding Author Michael Feldbrügge [email protected] orcid.org/0000-0003-0046-983X Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Kathi Zarnack Corresponding Author Kathi Zarnack [email protected] orcid.org/0000-0003-3527-3378 Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Author Information Lilli Olgeiser1, Carl Haag1, Susan Boerner2, Jernej Ule3,4, Anke Busch5, Janine Koepke6, Julian König5, Michael Feldbrügge *,1 and Kathi Zarnack *,2 1Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 2Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany 3The Francis Crick Institute, London, UK 4Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 5Institute of Molecular Biology gGmbH, Mainz, Germany 6Medical Clinic II (Molecular Pneumology), Excellence Cluster Cardio-Pulmonary System, Justus Liebig University of Gießen, Gießen, Germany *Corresponding author. Tel: +49 211 81 15475; E-mail: [email protected] *Corresponding author. Tel:+49 69 798 42506; E-mail: [email protected] EMBO Reports (2019)20:e46588https://doi.org/10.15252/embr.201846588 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3′ untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs. Synopsis A comparative iCLIP analysis of two endosomal RNA-binding proteins (RBPs) revealed that both proteins preferably bind conjointly in the 3′ UTR of target mRNAs. The key RBP Rrm4 binds distinct targets at landmark sites of translation, suggesting an intimate link between transport and translational regulation of cargo mRNAs. The small glycine-rich RNA binding protein Grp1 is a novel accessory component of endosomal transport mRNPs. The key RNA transport protein Rrm4 binds the motif UAUG via its third RRM domain and recognizes landmarks of translational regulation such as stop codons. Comparative iCLIP provides a first transcriptome-wide view of an endosomal mRNA transport machinery. Introduction All eukaryotic cells must accurately regulate the expression of proteins in time and space. To this end, many mRNAs accumulate at specific subcellular sites, and their local translation is exactly timed 123. mRNA localisation is achieved most commonly by active motor-dependent transport along the cytoskeleton. Functional transport units are messenger ribonucleoprotein complexes (mRNPs), consisting of various RNA-binding proteins (RBPs), accessory proteins and cargo mRNAs. Key factors are RBPs that recognise localisation elements (LEs) within mRNAs. For movement, the RBPs either interact with motors directly or are connected via linker proteins 14. We discovered co-transport of mRNPs on the cytoplasmic surface of early endosomes as a novel translocation mechanism of cargo mRNAs during hyphal growth in fungi 56. These endosomes shuttle along microtubules by the concerted action of plus-end-directed kinesin and minus-end-directed dynein 78. They serve as multipurpose platforms functioning not only during endocytic recycling but also during long-distance transport of whole organelles such as peroxisomes 9101112. Endosomal mRNA transport was uncovered analysing the RBP Rrm4 in the dimorphic phytopathogenic fungus Ustilago maydis (Fig EV1A) 1314. Loss of Rrm4 has no effects on the yeast form of the fungus. However, the absence of Rrm4 causes characteristic defects in unipolar growth when switching to the hyphal form: the fraction of bipolarly growing hyphae increases and the insertion of basal septa is delayed 615. In line with endosomal mRNA transport, Rrm4 binds mRNAs and shuttles on early endosomes along microtubules in vivo 516. Using the poly(A)-binding protein Pab1 as an mRNA marker revealed that loss of Rrm4 abolishes this transport, resulting in a gradient of mRNAs declining from the nucleus towards the cell periphery 17. Thus, one function of Rrm4 might be the general distribution of mRNAs within hyphae 17. Click here to expand this figure. Figure EV1. Loss of Grp1 causes defects in cell growth Hyphal form (6 hours post-induction, h.p.i.) of laboratory strain AB33 expressing a Gfp-tagged protein with nuclear localisation signal to stain the nucleus (N; λN-NLS-Gfp, phage protein λN fused to triple Gfp, containing a nuclear localisation signal; inverted fluorescence image shown; scale bar, 10 μm). Hyphae expand at the apical pole (arrow) and insert septa (asterisks) at the basal pole in regular time intervals resulting in the formation of empty sections. Schematic representation of the domain architecture of four small glycine-rich proteins (RRM, RNA recognition motif, green; GQ/R, glycine-rich region with arginine or glutamine, red). UmGrp1 from U. maydis (UMAG_02412), AtGRP7 from Arabidopsis thaliana (RBG7; NC_003071.7), HsRBM3 and HsCIRBP from Homo sapiens (NC_000023.11 and NC_000019.10, respectively). Number of amino acids indicated on the right. Results of preliminary affinity purification experiments using Rrm4-GfpTT as bait (see 4). Proteins with a functional link to Rrm4 are marked in red (this study) 1864. Peptide count: number of identified peptides corresponding to predicted protein; total peptide score: sum of all peptide scores corresponding to predicted protein, excluding the scores of duplicate matches; best peptide score: best score from all identified peptides corresponding to predicted protein. Note that the difference between total peptide score and best peptide score is a correction of the software depending on how many possible predicted candidates match to the identified peptide mass. Tandem affinity purification using Rrm4-GfpTT as bait. Protein bands were stained with Coomassie blue after SDS–PAGE. Proteins in boxed areas were identified as Rrm4 and Grp1 (size of marker proteins in kDa on the right). Growth curve of indicated AB33 derivatives growing in liquid culture. Data points represent averages from three independent experiments (n = 3). Error bars show s.d. Differential interference contrast (DIC) images of AB33 derivatives as yeast-like budding cells (scale bar, 10 μm). Length of budding cells (shown are merged data from three independent experiments, n = 3; > 100 cells per strain were analysed, wt, 269; rrm4Δ, 122 (only two independent experiments); grp1Δ, 263; grp1G, 318), overlaid with the mean of means, red line and s.e.m.; paired two-tailed Student's t-test on the mean cell lengths from the replicate experiments. Colonies of indicated AB33 strains grown in the yeast form incubated at different temperatures (28°C for 1 day or 16°C and 21°C for 5 days). Colonies of indicated AB33 strains grown in the yeast form. Incubated plates contained cell wall inhibitors (CM, complete medium for 1 day; CFW, 50 μM calcofluor-white for 4 days; CR, 57.4 μM Congo red for 4 days). Fluorescence images of the basal pole of hyphae of AB33 derivatives (6 h.p.i.). Septa (asterisks) were stained with CFW. White bars indicate exemplary length measurements of empty sections shown in Fig 1D (scale bar, 10 μm). Download figure Download PowerPoint Initial CLIP experiments with Rrm4 identified target mRNAs encoding chitinase Cts1 and septin Cdc3, among others 1718. The subcellular localisation of both proteins was Rrm4-dependent: loss of Rrm4 strongly reduced the secretion of the chitinase Cts1. Moreover, shuttling of the Cdc3 protein on early endosomes was abolished, and the gradient of septin filaments at the growth pole of hyphae was no longer formed 6. Since cdc3 mRNA and its encoded protein are found together with ribosomes on the same shuttling endosomes, we hypothesised that endosome-coupled translation of cdc3 mRNA during long-distance transport is critical for the efficient formation of septin filaments at the growth pole 6. This was supported by demonstrating that all four septin-encoding mRNAs are present on endosomes and that septin proteins assemble into heteromeric complexes on the cytoplasmic face of endosomes during long-distance transport 19. Thus, Rrm4-dependent mRNA transport regulates the specific localisation of the corresponding translation products. To understand this complex process at the transcriptome-wide level, we present herein an in vivo snapshot of RNA binding sites of endosomal RBPs on cargo mRNAs at single nucleotide resolution. Results Loss of the glycine-/glutamine-rich protein Grp1 affects hyphal growth In order to identify additional protein components involved in endosomal mRNA transport, we performed pilot affinity tag purification using Rrm4 as bait. We identified the potential RBP glycine-rich protein 1 (Grp1; UMAG_02412), which carries an N-terminal RNA recognition motif (RRM) domain followed by a short C-terminal region rich in glycine and glutamine (GQ-rich; Figs 1A and EV1B–D). The protein was similar to other small RRM proteins, such as human CIRBP or RBM3 and plant RBG7 (AtGRP7), all previously described as global stress regulators (Fig 1A) 2021. Figure 1. Grp1 is important for hyphal growth under suboptimal conditions Sequence alignment of glycine-rich proteins (Fig EV1B). UmGrp1 from U. maydis (UMAG_02412), AtGRP7 (RBG7) from A. thaliana (NC_003071.7), HsRBM3 and HsCIRBP from H. sapiens (NC_000023.11 and NC_000019.10, respectively). Amino acid positions within RRM that are identical in at least three proteins are highlighted in green (boxes indicate RNA contact regions RNP1 and RNP2). Glycine and arginine/glutamine residues in the glycine-rich region are labelled in red and blue, respectively. Hyphae of AB33 derivatives (6 h.p.i.). Growth direction and basal septa are marked by arrows and asterisks, respectively (scale bar, 10 μm). Hyphal length over time. Black and grey dots represent hyphae with and without septa, respectively. Shown are merged data (> 200 hyphae per strain) from three independent experiments, overlaid with the mean of means, red line and standard error of the mean (s.e.m.). Significance was assessed using paired two-tailed Student's t-test on the mean hyphal lengths from the replicate experiments, followed by multiple testing correction (Benjamini–Hochberg). Significant P-values (P < 0.05) for comparison against wild type within each time point are indicated above. Length of empty sections (see Fig EV1J). Merged data from three independent experiments are shown together, overlaid with mean of means, red line and s.e.m. (total hyphae analysed: wt, 250; grp1Δ, 190; grp1-gfp, 332; difference in means between wt and grp1-gfp was statistically not significant, ns, P = 0.95; paired two-tailed Student's t-test on the mean lengths from the replicate experiments (n = 3). Differential interference contrast (DIC, top) and fluorescence images (bottom) of AB33 hyphae (5 h.p.i.) stressed at 3 h.p.i. with cell wall inhibitor CFW (2.5 μM). Arrowheads indicate aberrant cell wall deformation (scale bar, 10 μm). Percentage of hyphae with normal cell walls with (stressed) and without (unstressed) CFW treatment (data points represent percentages of three independent experiments, n = 3; mean, dark grey lines and standard error of the mean, s.e.m., > 100 hyphae analysed per experiment; paired two-tailed Student's t-test on the means). Download figure Download PowerPoint For functional analysis, we generated deletion mutants in laboratory strain AB33. In this strain, the master transcription factor controlling hyphal growth is under control of an inducible promoter. Thus, hyphal growth can be elicited synchronously by changing the nitrogen source in the medium. The corresponding hyphae grow like wild type by tip expansion at the apical pole, while the nucleus is positioned in the centre and septa are inserted in regular intervals at the basal pole (Fig EV1A) 22. In the yeast form of AB33, we observed that loss of Grp1 resulted in slower proliferation as well as increased cell size (Fig EV1E–G). At lower temperatures, growth of the grp1Δ strain was affected even more strongly and it exhibited an altered colony morphology (Fig EV1H). This was consistent with a potential function in cold stress response, similar to the plant and human orthologues 2021. Furthermore, colony growth of the grp1Δ strain was strongly reduced upon treatment with inhibitors of cell wall biosynthesis, such as calcofluor-white (CFW) or Congo red (CR) 2324. Hence, loss of Grp1 might cause defects in cell wall formation (Fig EV1I). Studying hyphal growth revealed that, unlike observed in rrm4Δ strains, loss of Grp1 did not cause an increased amount of bipolar cells as it is characteristic for defects in microtubule-dependent transport (see rrm4Δ hyphae for comparison; Fig 1B and C) 12. On the contrary, under optimal growth conditions hyphae were significantly longer (Fig 1B and C), and the length of empty sections at the basal pole was increased (Figs 1D and EV1J). Hence, the coordination of hyphal growth may be disturbed in the absence of Grp1. In order to further investigate this, we stressed hyphae 3 hours post-induction (h.p.i.) of hyphal growth with the cell wall inhibitor CFW. In comparison with wild type hyphae, we observed a strongly increased number of grp1Δ hyphae with abnormal shapes (86%), indicating that cell wall integrity might be affected (Fig 1E and F). In summary, loss of Grp1 affects both yeast-like and hyphal growth. During the latter, Grp1 seems to be crucial for the correct coordination of cell wall expansion, which becomes particularly apparent during stress conditions. Grp1 is a novel component of endosomal mRNA transport To analyse the subcellular localisation of Grp1, we generated AB33 strains expressing Grp1 fused at its C-terminus to Gfp by homologous recombination. The functional Grp1-Gfp version accumulated in the cytoplasm as well as in the nucleus of hyphae. In comparison, the poly(A)-binding protein Pab1-Gfp was absent from the nucleus, suggesting that this localisation pattern is specific for Grp1 (Fig 2A). Importantly, a subpopulation of Grp1-Gfp moved bi-directionally in the cytoplasm with a velocity comparable to Rrm4-Gfp and Pab1-Gfp, which are known to shuttle on early endosomes (Fig 2A and B; Video EV1 ). Figure 2. Grp1 shuttles on Rrm4-positive endosomes throughout hyphae Micrographs (DIC and inverted fluorescence image; scale bar, 10 μm) and corresponding kymographs of AB33 hyphae (6 h.p.i.) expressing Grp1-Gfp, Rrm4-Gfp or Pab1-Gfp (arrow length on the left and bottom indicates time and distance, 10 s and 10 μm, respectively). To visualise directed movement of signals (distance over time) within a series of images, kymographs were generated by plotting the position of signals along a defined path (x-axis) for each frame of the corresponding video (y-axis). Bidirectional movement is visible as diagonal lines (yellow arrowheads; N, nucleus; Video EV1). For an example image of a complete AB33 hypha, see Fig EV1A. Average velocity of fluorescent signals per hypha for strains shown in (A) (movement of tracks with > 5 μm was scored as processive). Data points represent averages from three independent experiments (n = 3), with mean, red line and s.e.m. At least 20 signals/hyphae were analysed out of 12 hyphae per experiment (ns; P = 0.18 and P = 0.23) using a paired two-tailed Student's t-test on the means. Kymographs of hyphae of AB33 derivatives (6 h.p.i.) expressing pairs of red and green fluorescent proteins as indicated. Fluorescence signals were detected simultaneously using dual-view technology (arrow length as in A). Processive co-localising signals are marked by yellow arrowheads (Videos EV2, EV3 and EV4). Percentage of red fluorescent signals exhibiting co-localisation with the green fluorescent signal for strains shown in (C). Data points represent observed co-localisation in three independent experiments, mean, dark grey line and s.e.m. (n = 3, 11 hyphae each; paired two-tailed Student's t-test on the means; ns; P = 0.63 and P = 0.5). Kymographs comparing hyphae of AB33 derivatives (6 h.p.i.) expressing Rrm4-Gfp in the wild type (left) or grp1Δ strains (right) (processive signals marked by yellow arrowheads; arrow length on the left and bottom indicates time and distance, 10 s and 10 μm, respectively; Video EV5). Average velocity of fluorescent signals per hyphae for strains shown in (E) (movement of tracks with > 5 μm were scored as processive). Data points represent averages from three independent experiments (n = 3) with mean, black line and s.e.m. At least 20 signals/hypha were analysed out of at least 10 hyphae per experiment (ns; P = 0.27 and P = 0.4) using a paired two-tailed Student's t-test on the means. Kymographs comparing hyphae (6 h.p.i.) expressing Grp1-Gfp or Pab1-Gfp in wild type (left) with rrm4Δ strains (right; processive signals marked by yellow arrowheads; arrow length as in A). Hyphal tips (4 h.p.i.) of AB33 derivatives expressing Grp1-Gfp, Pab1-Gfp or Gfp alone comparing wild type (top) with rrm4Δ strains (bottom). Fluorescence micrographs in false colours (black/blue to red/white, low to high intensities, respectively; scale bar, 10 μm; ROI1 and ROI2-labelled circles exemplarily indicate regions of interest analysed in E). Ratio of signal intensities in strains shown in (H) comparing Gfp fluorescence at the tip (ROI1) and in close vicinity to the nucleus (ROI2; see 4). Bars represent mean and s.e.m. (wt: n = 160; rrm4Δ: n = 152), Pab1G (n = 126; rrm4Δ: n = 170), Gfp (n = 152; rrm4Δ: n = 210), unpaired two-tailed Student's t-test. Kymographs of hyphae of AB33 derivatives (6 h.p.i.) expressing pairs of red and green fluorescent proteins as indicated (arrow length as in A; Videos EV6 and EV7). Fluorescence signals were detected simultaneously using dual-view technology. Processive co-localising signals are marked by yellow arrowheads. Note that processive movement is completely lost in the lower panels. Only static signals, visualised as vertical lines, are remaining. Download figure Download PowerPoint To test whether Grp1 shuttles on Rrm4-positive endosomes, we performed dynamic co-localisation studies using dual-view technology 25. We generated AB33 strains co-expressing Grp1-Gfp and Rrm4 fused C-terminally to the red fluorescent protein tag-Rfp (tRfp) 26. For comparison, we used a strain expressing Pab1 fused to the red fluorescent protein mCherry 1727. Analysing hyphae 6 h.p.i. 99% of processive Grp1-Gfp signals co-migrated with Rrm4-tRfp, revealing extensive co-localisation of both proteins in shuttling units (Fig 2C and D; Video EV2). Consistently, 97% of processive Grp1-Gfp signals co-migrated with Pab1-mCherry, indicating that Grp1, like Pab1, was present on Rrm4-positive endosomes (Fig 2C and D; Videos EV3 and EV4). Thus, Grp1 appears to be a novel component of endosomal mRNPs that might already be recruited to transport mRNPs in the nucleus. The endosomal localisation of Grp1 depends on Rrm4 To investigate whether Grp1 has an influence on the shuttling of Rrm4-positive endosomes, we studied Rrm4 movement in grp1Δ strains. Loss of Grp1 altered neither processive Rrm4-Gfp movement nor the velocity of the respective endosomes (Fig 2E and F; Video EV5). Vice versa, studying Grp1-Gfp movement in the absence of Rrm4 revealed that its endosomal localisation depended on Rrm4 (Fig 2G). Importantly, similar to Pab1-Gfp, a gradient of Grp1-Gfp was formed in rrm4Δ hyphae, with a decreasing signal intensity towards the growing apex (Fig 2H and I) 17. Similar to Pab1, which is expected to associate with almost all poly(A) tails of mRNAs 28, Grp1 might therefore be distributed in association with many mRNAs (see below). To test whether Grp1-Gfp binds to endosomes in an mRNA-dependent manner, we generated AB33 strains expressing Rrm4mR123-tRfp. This Rrm4 variant carried point mutations in the RNP1 regions of RRM domains 1–3 causing a reduced RNA binding activity and loss of function of Rrm4 16. In dual-view experiments, we observed that Grp1-Gfp like Pab1-Gfp no longer shuttled in the presence of Rrm4mR123-tRfp (Fig 2J). Thus, the localisation of Grp1 depends on the presence of functional Rrm4, more precisely on its capability to bind RNA. In summary, we identified Grp1 as a novel component of endosomal mRNPs whose shuttling on Rrm4-positive endosomes depended on Rrm4 and mRNA. Rrm4 and Grp1 share thousands of target transcripts In order to learn more about the function of the two endosomal mRNP components Rrm4 and Grp1 during hyphal growth, we performed a comparative transcriptome-wide analysis of their RNA binding behaviour using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) 29. For application with fungal RBPs, we had to modify a number of steps in the iCLIP protocol (Fig EV2A–D; see 4) 30. One major challenge was the high RNase and protease activity in fungal cell extracts that resulted in a low yield of crosslinked protein-RNA complexes and short mRNA fragments. The most critical changes to the protocol came with the fast processing of crosslinked material and the identification of the optimal UV-C irradiation dose (Fig EV2B). Click here to expand this figure. Figure EV2. Improving the iCLIP protocol for fungal RBPs Grp1-Gfp/RNA complexes were size-separated on denaturing PAGE after UV-C irradiation and transferred to a nitrocellulose membrane (left). RNA was radioactively labelled, and protein-RNA complexes with covalently linked RNAs of different sizes were visible as smear above the expected molecular weight of the Grp1-Gfp protein (45 kDa; marked by arrowhead). RNA of four different regions of the membrane (A–D indicated on the right) were isolated from the membrane and were size-separated on a denaturing gel (6%) (right; nucleotide size marker on the left, bp). Autoradiographs showing Rrm4-Gfp, Grp1-Gfp and Gfp in complex with RNA after UV-C irradiation at 0, 160, 320, 480 and 640 mJ/cm2. Corresponding Western blots using anti-Gfp are shown below. Arrowheads indicate the expected molecular weight of the proteins (Rrm4-Gfp, 112 kDa; Grp1-Gfp, 45 kDa; Gfp, 27 kDa). After each irradiation step, the cells were mixed. Note that increased UV-C irradiation in combination with slow processing due to long time intervals was particularly harmful to the Rrm4 protein, which was completely degraded after four minutes of UV-C irradiation. Putative degradation products are marked by asterisks. Autoradiographs showing Rrm4-Gfp, Grp1-Gfp and Gfp in complex with RNA after single UV-C irradiation at 0, 100, 200, 300 or 400 mJ/cm². This time, mixing breaks were omitted and cells were harvested as quickly as possible. Corresponding Western blots are shown below. Labelling as above. We chose 200 mJ/cm2 as optimal UV-C irradiation dose, since the amount of unspecific Gfp-RNA complexes increased at higher doses. Arrowheads and asterisks as in (B). Amplification of the Rrm4-, Grp1- and Gfp-derived cDNA libraries with different numbers of PCR cycles (between 18 and 24; ctrl, control without template cDNA). The PCR products were separated on a native gel (6%) and stained with SYBR green I (nucleotide size marker on the left, bp). The size of the cDNA insert together with the adapters (cDNA insert = 20–30 nt; L3 adapter, RT-primer and P3/P5 Solexa primers = 128 nt) is expected to be ˜ 150–160 nt after amplification. Download figure Download PowerPoint Using the improved protocol, we found that Rrm4-Gfp and Grp1-Gfp displayed substantial crosslinking to RNA in vivo (compared to Gfp control; Fig 3A). As expected, the RNA signal was dependent on UV-C irradiation and sensitive to RNase I digestion. Upon iCLIP library preparation, we obtained more than 100 million sequencing reads, corresponding to 4.7 × 106 and 14.8 × 106 crosslink events for Rrm4 and Grp1, respectively (Fig EV3A and B). Reproducibility between two replicate experiments was high for both proteins, demonstrating the quality of the obtained dataset (Pearson correlation coefficient > 0.96, P-value < 2.22e-16; Fig EV3C). Figure 3. Rrm4 and Grp1 bind to thousands of target transcripts Autoradiograph and Western blot analyses for representative iCLIP experiments with Rrm4-Gfp, Grp1-Gfp and Gfp. Upper part: Upon radioactive labelling of co-purified RNAs, the protein-RNA complexes were size-separated in a denaturing polyacrylamide gel. Protein-RNA complexes are visible as smear above the size of the protein (Rrm4-Gfp, 112 kDa; Grp1-Gfp, 45 kDa; Gfp, 27 kDa; indicated by arrowheads on the right). Samples with and without UV-C irradiation and RNase I (see 4) are shown. Lower part: corresponding Western blot analysis using α-Gfp antibody (arrowheads and asterisks indicate expected protein sizes and putative degradation products, respectively). Summary of binding sites and target transcripts of Rrm4 and Grp1 (top). Venn diagram (below) illustrates the overlap of Rrm4 and Grp1 target transcripts. iCLIP data for Rrm4 and Grp1 on cdc3 (UMAG_10503; crosslink events per nucleotide from two experimental replicates [light grey/light blue] and merged data [grey/blue] from AB33 filaments, 6 h.p.i.). Track below the merged iCLIP data shows binding sites for each protein (bs, red). Note that crosslink events in the 5′ UTR and first exon of cdc3 were not assigned as binding sites due to low reprodu
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